Cis Regulatory Motifs in Adult Articular Chondroytes

描述(申请人提供)：在2003年资助的最初的RFA(关节炎、肌肉骨骼和皮肤疾病的基因表达研究)中，对成人关节软骨细胞进行了基因表达研究，建立了一组共调控基因，并对这些基因进行了计算分析，以寻找共调控基序。由此发现了一条与软骨细胞代谢有关的新的转录途径：导致转录因子类固醇调节元件结合蛋白(SREBPs)产生的途径。在这项拨款续期提案中，我们描述了将我们的工作重点放在这一途径上的计划，研究组织特异性敲除酶位点-1蛋白所产生的表型，这是一种激活SREBPs所必需的酶。1位点蛋白水解酶的突变或基因敲除会导致严重的软骨发育不良，没有软骨内骨形成。我们的结果表明，Site-1蛋白酶除了在SREBP途径中的功能外，可能在软骨形成中具有独立于其激活转录因子的能力的功能。这些研究为软骨研究提供了一个全新的方向，并有可能影响软骨基因的转录调控、软骨内骨的形成和关节软骨的修复。因此，特异性AIM 1旨在研究Site-1蛋白在体内软骨形成和维持软骨细胞表型中的作用，而特异性AIM 2旨在从分子水平探讨这一表型在体外的作用机制。已经制造了软骨特异性基因敲除小鼠，这些小鼠患有严重的软骨发育不良，没有软骨内骨形成。将通过分析软骨特异性基因敲除和其他基因敲除表型，包括生长板中更集中的基因敲除和诱导性基因敲除系统，来确定缺陷的机制。体外研究将包括整个胫骨的器官培养，细胞生物合成机制的分析，以及软骨细胞外基质的分析。

DESCRIPTION (provided by applicant): In the original RFA (Gene Expression Studies in Arthritis and Musculoskeletal and Skin Diseases), funded in 2003, gene expression studies were conducted in adult articular chondrocytes, sets of co-regulated genes were established and these genes were computationally analyzed for co-regulatory motifs. Arising from this proposal was the discovery of a new transcriptional pathway involved in chondrocyte metabolism: the pathway leading to the generation of the transcription factors steroid regulatory element binding proteins (SREBPs). In this grant renewal proposal, we describe plans to focus our work on this pathway, studying the phenotype generated by tissue-specific knock out of the enzyme Site-1 Protease, an enzyme necessary for activation of SREBPs. Mutation of Site-1 protease or knock-out of the gene results in a severe chondrodysplasia with no endochondral bone formation. Our results point to the possibility that Site-1 protease, in addition to its function in the SREBP pathway, may have a function in chondrogenesis that is independent of its ability to activate the transcription factor. These studies provide an entirely new direction in cartilage research and have the potential to impact both the transcriptional regulation of cartilage genes, the formation of endochondral bone and articular cartilage repair. Therefore, SPECIFIC AIM 1 is designed to investigate the role of Site-1 protease in chondrogenesis and in maintenance of the chondrocyte phenotype in vivo while SPECIFIC AIM 2 is designed to pursue the mechanism of this phenotype on a molecular basis in vitro. Cartilage-specific knock out mice have been made that have severe chondrodysplasia and no endochondral bone formation. Studies will be undertaken to determine the mechanism of the defect by analysis of this cartilage-specific knockout and other knock-out phenotypes, including more focused knock out in the growth plate and an inducible knock out system. In vitro studies will include organ culture of whole tibias, analysis of biosynthetic mechanisms of the cells and analysis of the extracellular matrix of the cartilage.